The long-term objective of this research project is to provide experimental evidence for the existence of a novel GTP-binding protein that appears to act as a membrane transducer coupling certain cell-surface receptors to the enzyme phospholipase C. Activation of phospholipase C results in the formation of the second messenger inositol trisphosphate (IP3) which, in turn, stimulates the release of calcium from intracellular store. An elevation of intracellular calcium then triggers the appropriate cellular response such as neurotransmitter or hormone release or smooth muscle contraction depending on the cell type. It is proposed that the putative GTP binding protein linking the cell- surface receptor to phospholipase C plays a crucial initial step in the response of a cell to an agonist. The specific aims of this proposal are to determine the following: (1) the requirement of guanine nucleotides for TRH receptor stimulation of IP3 formation in lysates of 7315c cells will be established; the ability of GTP, GTP gamma S, GDP and GDPbetaS to enhance or inhibit TRH stimulation of IP3 will be investigated; (2) the ability of TRH to stimulate GTPase will be tested; (3) the ability of guanine nucleotides to affect agonist binding will be tested; (4) as a first step toward isolating the TRH receptor and its GTP binding protein, optimal conditions for solubilizing an active and GTP- sensitive TRH receptor will be determined; (5) an attempt will be made to isolate the TRH receptor (in close association with its GTP binding protein) on a wheat germ lectin column followed by chromatography on a TRH-agonist affinity column, (6) if the TRH receptor and its GTP binding protein are successfully isolated, an attempt will be made to reconstitute them in phospholipid vesicles; the interaction of the receptor with its GTP binding protein will be evidenced by the ability of TRH to stimulate GTPase activity in this preparation, and (7) a number of bacterial toxins will be tested for their ability to catalyze the ADP ribosylation of the GTP-binding protein associated with the TRH receptor.